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Article

Hard Seed Characteristics and Seed Vigor of Ormosia hosiei

by
Li Dai
,
Yanwei Chen
and
Xiaoli Wei
*
College of Forest, Guizhou University, Guiyang 550025, China
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(5), 1077; https://doi.org/10.3390/agriculture13051077
Submission received: 24 March 2023 / Revised: 13 May 2023 / Accepted: 16 May 2023 / Published: 18 May 2023
(This article belongs to the Section Seed Science and Technology)
Browse Figures
Versions Notes

Abstract

:
The Ormosia hosiei is a secondary protected wild plant in China, and its seed has a hardness rate of 86%. To explore the characteristics of the hard seed and the difference in seed vigor between hard seeds and the non-hard seeds of O. hosiei, the water absorption characteristics, germination characteristics, seed coat structure, seed coat permeability, enzyme activity, and main biochemical substances of the O. hosiei seeds were determined. The results showed that the hard and dense seed coat is the main obstacle to absorbing water of O. hosiei seeds; the main reason is that the seed coat is composed of cuticle, palisade cells, and thick-walled cells with impermeability. Hot water treatment can soften the seed coat and remove wax and grease from the seed coat, and concentrated sulfuric acid treatment can corrode the local seed coat and break the barrier of cuticle and palisade tissue. The effectiveness of concentrated sulfuric acid to break the dormancy of O. hosiei hard seeds is better than that of hot water treatment, but the damage to the seed coat is larger and irreversible. The germination, germination index, root activity, TTCH content, and SOD activity of the hard seeds were higher than those of the non-hard seeds, but the SSC, conductivity value, and MDA content were lower than those of the non-hard seeds.

1. Introduction

Hard seededness is common in plants, most commonly found in the seeds of legumes [1,2]. It is generally believed that hard seeds can germinate normally after pretreatment [3]; however, due to the poor water permeability of the seed coat [4], it is difficult to break the barrier of a hard seed coat only by water absorption under natural conditions [5]. This causes many mature seeds to fail to germinate even under appropriate germination conditions [6], directly affecting the seed germination rate. This brings inconvenience to seed quality inspection and sowing and to seedling raising in production [7,8]. Therefore, the causes of hard seed formation, the influencing factors, and breaking the hard method have been hot topics of research for scholars.
Plant seed coat cell level and arrangement, pigment type, and content, as well as impermeable material deposition, etc. result in high mechanical strength and poor air and water permeability of the seed coat, limiting embryo expansion and forming hard seeds [9,10]. The seed coat of most hard seeds is extremely tough, mainly because the seed coat has a well-developed stratum corneum, widely developed palisade cells and bone stone cells [11], and long and narrowly shaped palisade cells that are closely arranged and accumulate a large amount of calcium salt or silicate [12]. This causes the inner wall of the seed coat to be thickened and the pores blocked, meaning that the water and gas cannot be inhaled and exuded and the exchange between the inside and outside is not smooth; as a result, the biochemical reaction of seed germination cannot be carried out smoothly, and the embryo cannot grow normally. Factors affecting hard seed formation can be divided into internal and external factors [13,14]. Internal factors include plant species and seed morphology [15]. External factors mainly refer to environmental factors such as soil nutrient and water supply, air relative humidity, temperature and light during seed development, extreme drying, and storage [16]. Commonly, for a plant, there are several methods to remove hard seeds. Mechanical treatment refers to the physical abrasion damage to the hard seed coat, which enhances the water permeability and air permeability of seeds and can quickly remove the hard seed coat on the mechanical restriction of seeds. It can reduce the hard seed rate and promote seed germination [17]. It is the most effective way to treat hard seeds [18]. Many studies have found that pretreatment with hot water can soften hard seed coats and leach out the chemicals that inhibit seed germination [19], thereby increasing seed coat permeability. This is the most commonly used high-temperature treatment method. In addition, treatment with strong acid or a bleaching agent can corrode the seed coat so that the seed coat becomes thinner and can eliminate the plug holes and other parts, break seed dormancy, and significantly improve seed germination rate and germination potential [20,21].
Ormosia hosiei Hemsl. et Wils. is an important tree species of Leguminosae; it is listed as a national secondary wild protection plant in China. O. hosiei is mainly distributed in the Fujian, Sichuan, Jiangsu, Zhejiang, and Guizhou provinces in China. Its heartwood is hard and has a fine wood grain, which is not only valuable for wood use but also has high medicinal and horticultural value. As with most legume seeds, there is a clear hard seed phenomenon in the seed of O. hosiei. Due to the large seed and hard seed coat of O. hosiei, it is difficult for the seed to absorb water, which limits its germination, emergence, and seedling growth. Hence, if the seed is not pretreated, it will result in a low germination rate and irregular emergence, which brings difficulties to the seedling of O. hosiei. Previous studies on O. hosiei mainly involved tree and litter nutrients [22], population characteristics under different disturbance conditions [23], seedling resistance [24], etc. However, there are few reports on the hard characteristics of the seeds.
We studied the water absorption characteristics, germination characteristics, seed coat permeability, enzyme activity, and inclusion substances of the hard seed of O. hosiei, as well as the microstructure of the seed coat. Specifically, we would like to know the following questions: (i) What are the reasons for limiting water absorption of O. hosiei hard seeds? (ii) How do hot water treatment and acid etching enhance the water absorption capacity of hard seeds? (iii) What is the difference in seed germination between the hard seeds and non-hard seeds of O. hosiei?

2. Materials and Methods

2.1. Materials

The seeds of the O. hosiei were collected from Guanling County, Guizhou Province of China in November 2021, with a purity of 98%, a weight of 1029.7 g per 1000 grains, and a hardness rate of 86%.

2.1.1. Determination of Seed Water Absorption

The seeds of O. hosiei were divided into A and B groups. The A group of seeds was not treated, but the B group was treated with a knife to cut the seed coat, and 10 seeds per treatment were soaked in a beaker containing 50 mL of 45 °C water, stirred, and cooled; then, the seeds were removed every 12 h, the water on the surface of the seeds was sucked dry using the filter paper, and the seeds were weighed and recorded until constant weight.
Water absorption rate (%) = (weight after soaking − weight before soaking)/weight before soaking × 100

2.1.2. Hard Seed Sample Selection

The seeds of 5000 red bean species were selected from the pot, soaked in warm water with an initial temperature of 45 °C, and naturally cooled. The seeds that naturally imbibed on the first day were recorded as (X1), and the seeds that were not imbibed continued to be placed in the basin. The seeds that imbibed on the second to sixth days were recorded as X2, X3, X4, X5, and X6. At this time, the seeds continued to soak, and the ratio of imbibition seeds was less than 1%. Accordingly, the seeds that had not been imbibed on the sixth day were used as the hard seeds (H) with the highest degree of hardness in this experiment. The germination index, seed coat permeability, and biochemical index of X1, X2, X3, X4, X5, and H were determined. The hard seeds were treated with 98% concentrated sulfuric acid for 10 min and then soaked for 24 h at room temperature.

2.1.3. Sample Selection for Breaking Hard Seeds

The hard seeds (H) of 2.1.2 were selected for the following treatment. (1) H2SO4 treatment: the hard seeds were soaked in 98% H2SO4 for 5 min, 10 min, 15 min, and 20 min, respectively. (2) Hot water treatment: the hard seeds were soaked in beakers containing hot water at 70 °C, 80 °C, 90 °C, and 100 °C, respectively. Natural cooling occurred, with (3) replicates per treatment.

2.1.4. Electron Microscopic Scanning of Seed Coat Structures for Hard and Non-Solid Seeds

The hard seeds of O. hosiei were soaked in 25 °C water (scarified seeds), 100 °C hot water, and concentrated H2SO4 for 10 min, respectively. After washing and drying, the seed coat and seed coat section of each treatment sample were placed on the sample table in turn. After gold plating, their microstructure was observed under a scanning electron microscope, and the seeds without any treatment were used as control.

2.1.5. Germination Determination of Non-Solid Seeds and Hard Seeds

With 25 °C artificial climate box and filter paper germination bed, the imbibed seeds (X1–X6) obtained by 2.1.2 and the treated hard seeds (Hot water soaked 24 h at 100 °C, cooled naturally) were taken, and each treatment was repeated four times with 100 seeds per time. From the climate box, the germination of seeds was observed and recorded every day, and the corresponding indexes were measured. After germination, the seedlings were taken to determine the root activity, and the root activity was determined by triphenyltetrazolium chloride staining. The calculation formula for germination of each index is as follows:
Germination ratio (%) = (number of seeds germinated in the specified entire germination time/number of seeds to be tested) × 100
Germination potential (%) = (cumulative seed germination at peak/number of seeds for testing) × 100
Germination index: Gi = (Gt/Dt) (where Gi represents the germination index, Gt is the number of germinated grains on the day, and Dt is the corresponding number of days.)

2.1.6. Assay of Seed Coat Permeability

The steps of this process are as follows: take hard seed sample selection to obtain 30 seeds of imbibition at different times and 30 seeds of hard solid seeds after treatment. Perform each treatment of 10 seeds for 1 repeat, a total of 3 times, then to each add 50 mL of deionized water, placed at a constant temperature of 25 °C for 6 to 8 h, with DDS-2 type conductivity to determine the conductivity of the leaching fluid. Conductivity (µs/cm·g) = ((Repeat 1 Conductivity − Control Conductivity)/Sample Weight) + ((Repeat 2 Conductivity − Control Conductivity)/Sample Weight) + ((Repeat 3 Conductivity − Control Conductivity)/Sample Weight)

2.1.7. Determination of SSC, TTCH, SOD, and MDA

The imbibition seeds and hard seeds obtained at different times obtained in the determination of seed water absorption were taken as the materials to determine the main enzyme activity and biochemical content of the seeds. The determination of soluble sugar content (SSC) was based on the anthracene colorimetric method [25]. Dehydrogenase (TTCH) content was measured by staining of triphenyltetrazole chloride [26], superoxide dismutase (SOD) activity was determined by nitrogen blue tetrazole method [1], and malondialdehyde (MDA) content determination was determined by thiobarbituric acid method [21]. Each of the above parameters was measured in 3 biological replicates.

2.2. Statistical Analyses

Values presented are mean ± standard deviation (SD) of three replicates. SPSS 26.0 statistical software was used to analyze the data, and the difference in significance between the mean values was determined at the probability levels of 0.05 and 0.01 using the one-way ANOVA and the least significant difference method.

3. Results

3.1. Water Absorption Characteristics of the Seeds of O. hosiei

The water absorption of O. hosiei seeds showed a trend of increasing first and then stabilizing over time (Figure 1). The water absorption of intact seeds (A) was only 17% after soaking for 12 h, then the water absorption rate increased slowly, tended to be stable after 60 h, and reached a peak of 56% after 96 h. However, the water absorption rate of the scarified seeds (B) of O. hosiei reached 108% after soaking for 12 h, the water absorption rate continued to increase within 48 h, and the peak water absorption rate of 84 h was 203%, reaching saturation, which was 147% higher than that of the intact seed (A). The water absorption rate of the intact seeds (A) and scarified seeds (B) of O. hosiei was significantly different (p < 0.05). It can be seen that the hard and dense seed coat is the main obstacle to the imbibition of O. hosiei seeds.

3.2. Electron Microscopy Results of Seed Coat

The results of electron microscopy showed that the seed coat treated with hot water and concentrated sulfuric acid changed greatly compared with untreated (Figure 2 and Figure 3). As shown in Figure 2A, the surface of the O. hosiei hard seed coat is covered with one or more layers of glossy, unevenly distributed, hydrophobic, irregularly scaly wax and hard and dense cuticle. After hot water treatment and concentrated sulfuric acid treatment, there was little residual wax on the surface of the hard seed coat of O. hosiei, and the seeds treated with hot water softened. The surface of the seed coat showed mild shrinkage and high shrinkage, and there were strips of large and small pores (Figure 2B). Large circular holes appeared on the surface of the seed coat treated with concentrated sulfuric acid (Figure 2C).
The coat of the O. hosiei seed is composed of a surface layer, outer layer, and inner layer (Figure 3A–C). The surface layer is a thicker cuticle with impermeability. The outer layer is composed of palisade cells and thick-walled cell layers (the palisade layer was 295.15 μm thick). Immediately adjacent to the cuticle is a layer of longitudinal tightly arranged palisade cells. Below the palisade tissue is a layer of hard and woody sclerenchyma. The sclerenchyma is composed of stone cells, arranged closely and without intercellular space, so the water absorption of palisade cells and sclerenchyma is inferior. The inner layer is composed of parenchyma cells, which have an irregular shape, loose organization, and strong water absorption.
The seed coat thickness of hard seeds treated with hot water and concentrated sulfuric acid was higher than that of untreated seeds. With a scanning electron microscope, it was found that the increased thickness of the seed coat mainly manifested in the parenchyma cell of the seed coat. This is due to hot water treatment and concentrated sulfuric acid treatment leading to large cracks and holes in the palisade cells and thick-walled cell layers of the seed coat; the parenchyma cells layer could absorb water through these cracks and holes, so that the parenchyma cells layer became thicker. Further observation showed that compared with hot water treatment, the hard seeds treated with concentrated sulfuric acid had deep and wide cracks, which could make the parenchyma cells absorb water quickly in a short time. Therefore, the effect of concentrated sulfuric acid treatment to break the dormancy of O. hosiei hard seeds is better than that of hot water treatment, but the damage to the seed coat is larger and irreversible.

3.3. Comparison of Germination Indexes of Hard Seed by the Different Etching Treatments and Hot Water Treatments

The germination rate, germination potential, germination index, and seedling root activity of O. hosiei hard seeds treated with different acid etching increased first and then decreased with the increase in treatment time (Table 1). There was no significant difference in the germination rate of O. hosiei seeds etched for 5–15 min (p > 0.05), and the germination rate of seeds etched for 20 min was not significant. The germination rate of seeds treated with acid etching for 30 min was the lowest. In general, the germination rate of seeds treated with acid etching was significantly higher than that of the control (p < 0.01). The germination rate, germination potential, germination index, and seedling root activity of red bean seeds treated with acid etching for 20 min were compared. The control increased by 46%, 40%, 18%, and 19%. Subsequently, with the extension of the soaking time of concentrated H2SO4, the values of each index gradually decreased, indicating that the treatment time of concentrated H2SO4 was too short or too long to achieve the desired effect of H2SO4.
The germination rate, germination potential, germination index, and seedling root activity of O. hosiei hard seeds treated with hot water increased with the increase in treatment temperature (Table 1). The germination indexes of hard seeds treated at different temperatures were significantly higher than those of the control group (p < 0.01). The germination index and seedling root activity of hard seeds treated with hot water were significantly higher than those of the control, which effectively broke the dormancy of O. hosiei seeds. The germination index of hard seeds soaked in 100 °C hot water was the highest, which was significantly higher than that of hard seeds treated with 70–90 °C hot water. The skin of O. hosiei was hard and thick. High-temperature treatment could improve the water permeability of the seed coat, thereby increasing the germination rate of seeds and breaking the dormancy of hard seeds.

3.4. Comparison of Biochemical Substance Content and Seed Coat Permeability of Hard Seeds by the Different Etching Treatments and Hot Water Treatments

With the extension of acid etching time, the electrical conductivity of the hard seeds of O. hosiei generally increased (Table 2). The electrical conductivity of seeds treated with acid etching for 25 min and 30 min gradually increased, and the electrical conductivity of seeds treated with acid etching was significantly higher than CK (p < 0.01). The soluble sugar content decreased first and then increased. The soluble sugar content of seeds treated with acid etching for 20 min was the lowest, and the soluble sugar content of seeds treated with acid etching for 25–30 min was relatively high. The soluble sugar content of each treatment was significantly higher than CK (p < 0.01). The TTCH content decreased with the increase in temperature. The TTCH content of red bean seeds treated with acid etching for 10 min was the highest, and the TTCH content of seeds treated with acid etching for 25 min and 30 min was lower. The TTCH content of seeds treated with acid etching was significantly lower than CK (p < 0.05). The content of SOD increased first and then decreased with the increase in acid etching time. The content of SOD was the highest when the acid etching time was 20 min. The content of SOD in each acid etching treatment was significantly different (p > 0.05). The MDA content of each acid etching treatment was significantly higher than CK (p < 0.01).
The electrical conductivity of O. hosiei seeds increased with the increase in treatment temperature (Table 2). The seed conductivity index of 70–100 °C temperature treatment was very close, the difference was not significant (p > 0.05), and the temperature treatment was significantly higher than CK. The soluble sugar content decreased first and then increased with the increase in temperature. The soluble sugar content of O. hosiei treated at 70–100 °C was slightly higher than CK, and there was no significant difference between each treatment and the control (p > 0.05). The TTCH content decreased with the increase in temperature. The TTCH content of seeds treated at 70 °C was the highest. The TTCH content of seeds treated at each temperature was significantly higher than CK. The MDA content of seeds treated at each temperature was significantly higher than that of the control (p < 0.01).
The order of seed vigor of different treatments was high temperature 90 °C > high temperature 100 °C > high temperature 80 °C > high temperature 70 °C and etching > acid etching 20 min > acid etching 10 min > acid etching 15 min > acid etching 5 min > acid etching 25 min > acid etching 30 min, through the high temperature. The seed vigor of the processed O. hosiei was higher than that of the acid etching treatment.

3.5. Comparison of Germination Indexes of Non-Hard Seeds and Hard Seeds

It can be seen from Table 3 that the germination ratio, germination potential, germination index, and seedling root activity of the non-hard seeds of O. hosiei increased with the increase in the degree of hard seed, and the germination index and seedling root activity of hard seeds were significantly higher than those of non-hard seeds (p < 0.01). Daily germination rates can reflect the whole germination process of seeds with different hardness [21]. The germination ratio of X1 seeds was the lowest, only 24%, with the extension of imbibition time; the seed germination ratio increased day by day, and the germination rates of X4 and X5 seeds were close, but the difference was not significant (p > 0.05). The germination ratio, germination potential, germination index, and seedling root activity of hard seed were 51%, 53%, 400%, and 217% higher than those of the first-day-imbibed seeds.

3.6. Comparison of Seed Coat Permeability of Non-Hard Seeds and Hard Seeds

The level of electrical conductivity reflects the amount of cell leakage and is negatively correlated with seed vigor [22]. The electrical conductivity of O. hosiei seeds decreased with the increase in soaking time, and the electrical conductivity of the hard seeds was lower than that of the non-hard seeds (Figure 4). The electric conductivity of the seeds of X1–X6 was significantly higher than that of hard seeds (p < 0.01). The electrical conductivity of X1 was 179% higher than that of the hard seeds. The electrical conductivity of X2 and X3 seeds had no significant difference (p > 0.05). The electrical conductivity of X4–X6 seeds was 138%, 129%, and 110% higher than that of hard seeds, respectively.

3.7. Comparison of Biochemical Substance Content between Hard Seeds and Non-Hard Seeds

3.7.1. Soluble Sugar Content and Malondialdehyde Content

With the prolongation of soaking time, the soluble sugar content (SSC) and malondialdehyde (MDA) of the seeds of O. hosiei showed a declined trend (Figure 5A,B). The SSC of O. hosiei seeds decreased slowly in X1–X6, and X6 decreased by 198% compared with X1 (p < 0.01). The SSC of X1–X6 imbibition seeds was 338%, 300%, 162%, 138%, 129%, and 110% higher than that of O. hosiei hard seeds (p < 0.01), respectively. In X1–X6, the MDA in O. hosiei seeds decreased rapidly, reaching the lowest value in X6: X6 was 332% lower than X1, and the difference was extremely significant (p < 0.01). The MDA content of X1–X6 imbibed seeds was 631%, 580%, 465%, 352%, 312%, and 190% higher than that of the O. hosiei hard seeds, respectively. In conclusion, in the imbibition process of O. hosiei seeds, because non-hard seeds were more likely to deteriorate than hard seeds, the MDA content of the fast imbibition seeds was higher than that of the hard seeds.

3.7.2. Dehydrogenase Activity and Superoxide Dismutase Activity

As shown in Figure 5C,D, the activities of superoxide dismutase (SOD) and dehydrogenase (TTCH) in the seeds of X1–X6 increased with the prolongation of soaking time. In X1–X3, the TTCH content of imbibed seeds of O. hosiei changed little, and X4–X6 increased rapidly. X6 increased by 133% compared with X1, and the difference was extremely significant (p < 0.01). The SOD activity of X1–X6 seeds continued to increase with the soaking time and reached a peak in X6, which was 523% higher than that of X1. Furthermore, TTCH content and SOD activity of hard seeds for O. hosiei were extreme and significantly higher than those of non-hard seeds (p < 0.01). TTCH content in hard seeds increased by 199% compared with the average of non-hard seeds. The SOD activity of O. hosiei hard seeds was 822%, 663%, 354%, 326%, 265%, and 161% higher than that of X1–X6 seeds, respectively.

4. Discussion

4.1. Anatomical Structure and Water Absorption of O. hosiei Seeds

Most studies have shown that moisture is one of the main limiting factors for hard seed germination [27]. The rate of seed water absorption can reflect the level of seed hardness to some extent [28]. In this experiment, it was found that the hard seed of O. hosiei had difficulty absorbing water, but the water absorption and swelling abilities of the seeds were enhanced after the peeling treatment. The water absorption of the scarified O. hosiei seed was 147% higher than that of the intact seeds. Previous studies suggested that the initial water-absorbing site of some leguminous seeds after dormancy breaking was the ridge, while other seeds were other sites except raphe [29,30]. However, for the intact hard seeds, raphe water absorption was less, and the seed coats were hard, compact, and impervious, so the seed coat is the O. hosiei seed imbibition’s main obstacle.
The impermeability of the seed coat is usually related to the microstructure, as the arrangement and composition of the cells in the seed coat will directly affect the permeability of the seed [9,31]. In this study, we found that the seed coats of O. hosiei seeds were composed of cuticle, palisade cells, a thick-walled cell layer, and a parenchyma cell layer. Although the parenchyma cells with loose tissue had a strong water absorption ability, for the cuticle, palisade cells, and thick-walled cell layers with compact structures, the water absorption was poor. It is generally believed that the first barrier to prevent water from entering the seed is the dense cuticle layer on the surface of the seed coat, and the second barrier is the fence layer structure [9]. The palisade cells are long and narrow in shape, and the cells are arranged neatly and closely. They can also accumulate a large amount of calcium salts or silicates [4]. In addition, their cell walls are mainly composed of cellulose, lignin, cutin, protein, pectin, etc. [32], so that the cell walls have high strength and resistance to chemical degradation. These features affect the thickness and hardness of the seed coat and ultimately lead to poor water absorption of hard seeds.
In the palisade cell layer, some seeds also have the special structure of the light line, which is considered to be the most impenetrable area for seeds [12]. However, the presence of bright lines was not clearly observed in the scanning electron microscope images of O. hosiei. Previous studies have found that the hard seed coat surface usually contains suppository, lipids, wax, and other hydrophobic chemicals, which will also increase the difficulty of seed imbibition [33]. In this study, it was also found that the untreated hard seeds were densely covered with oval tumor-like waxes of different sizes on the surface of the seed coat. These waxes were hydrophobic and seriously hindered water from entering the seeds, which in turn affected the water absorption of O. hosiei.
Previous studies have found that hot water treatment can soften the hard seed coat [12]. After hot water treatment, the wax in the umbilicus of the seed will decrease or disappear [33] so that the seed can absorb water quickly. Concentrated sulfuric acid can corrode the local seed coat and eliminate the plug holes and other parts; by concentrated sulfuric acid treatment of seeds, water absorption will be significantly accelerated [34]. In this study, hot water treatment not only caused the seed coat of O. hosiei to shrink, crack, and soften but also removed the seed coat surface wax and oil and improved the permeability of the seed coat. Concentrated sulfuric acid treatment can destroy the seed coat structure, and with the extension of treatment time, the damage degree of the seed coat structure is aggravated, the cuticle structure is completely destroyed, and even some thick-walled tissues, palisade layers, and skeleton stone cells are corroded, so that the seed coats of O. hosiei seeds have holes of different sizes, which reduce the mechanical obstacles of the seed coat and break the barrier affecting the water absorption rate of the seed. Compared with concentrated sulfuric acid treatment, hot water treatment is indeed more economical and environmentally friendly, but its performance in breaking dormancy is not as fast and effective as concentrated sulfuric acid, which is greatly affected by treatment temperature and duration.
According to the results of the water absorption curve and electron microscope observation, however, the seed vigor of red bean seeds treated with high temperature was higher than that of acid etching treatment. The seed vigor of high-temperature treatment was the highest when soaked in hot water at 90 °C, and the seed vigor of acid etching treatment was the highest when soaked in concentrated H2SO4 for 20 min. Upon comprehensive comparison of 10 treatments, the high temperature 90 °C treatment had the best effect on breaking hard seeds and the highest seed vigor, while acid etching 30 min treatment had no obvious effect on breaking the hard seeds, had the lowest seed vigor, and even caused irreversible damage to the seeds [35]. Thus, the hard seeds of O. hosiei should be pretreated before sowing to break their dormancy.

4.2. Germination Ability and Seedling Root Activity of O. hosiei Seeds

Hard seeds are generally highly active and can germinate and sprout under suitable conditions [4,36,37]. Hard seeds have important ecological significance. Seed populations can germinate in different seasons or periods, and by reducing or preventing the risk of seed germination in adverse environments, the adaptability of plants is improved and the continuation of the population is ensured [9]. The vigor level of hard seeds is the key to measuring their ability to adapt to the environment. Seed vitality is the sum of seed germination and emergence rate, seedling growth potential, plant resistance, and production potential, and is an important index of seed quality. Thus, any single index cannot be used as a standard to evaluate the seed vigor; in judging the level of seed vigor, germination potential, germination rate, germination index, and seedling root activity must be tested and evaluated [38,39]. This study found that the germination rate, germination potential, germination index, and seedling root activity of the non-hard seeds of O. hosiei increased with the increase in seed hardness. The germination index and seedling root activity of hard seeds were extremely significantly higher than those of non-hard seeds (p < 0.01), and the seed vigor level was affected by the degree of hardness and increased with the increase in the degree of hardness. The vigor index of hard seeds was the highest, which is because the seed’s physiological maturity is at the maximum viability, but due to the effect of environmental factors, seed viability will continue to decline over storage time. Because the seed coat of hard seeds is hard and compact, it is less disturbed by the external environment, so the viability of hard seeds is higher than non-hard seeds.

4.3. Biochemical Character and Seed Vigor of the Hard Seeds of O. hosiei

The biochemical characteristics of seeds are closely related to seed vigor. Previous studies have shown that there are obvious differences in biochemical characteristics between hard seeds and non-hard seeds [40]. On one hand, the high permeability of the seed coat of the non-hard seed will make the seed imbibe rapidly in a short time, which not only causes physical damage to the seed but also causes membrane damage. The low-vigor seed is damaged by the membrane structure; as the permeability of the membrane is increased, the amount of electrolyte and soluble substance extravasation is greater, so the conductivity and soluble sugar content of the leachate are higher [41]. On the other hand, in the process of seed imbibition, the reactive oxygen entering the cells increases, which accelerates lipid peroxidation and reduces seed vigor. When seed vigor declines, a series of physiological and biochemical changes initially occur, followed by ultrastructural changes [5,40]. A large number of studies have found that the role of some enzymes in the seeds and the occurrence of automatic oxidation will produce free radicals, and free radicals will cause the seeds of macromolecular polymers, including membrane lipids, to be damaged [11]. In high-vigor seeds, the production and scavenging reactions of free radicals are balanced, but in lower-vigor seeds, enzyme passivation and protein denaturation will be produced due to insufficient clearance reaction. Superoxide dismutase as a specific scavenging antioxidant enzymes plays an important role in the free radical scavenging system, and the higher the degree of hard seeds, the higher the level of SOD activity during germination [42]. However, in the process of seed imbibition, SOD activity will decrease, the ability to scavenge free radicals and peroxides will be weakened, and free radicals will continue to accumulate, attacking membrane lipid molecules, causing peroxidation, and forming organic free radicals. Moreover, organic free radicals attack other fatty acid chains and protein molecules and further oxidize themselves into the final product of malondialdehyde (MDA), etc. [25,43]. MDA can, in turn, inhibit the activity of cytoprotective enzymes and reduce the content of antioxidants; the cells have a significant toxic effect [44]. Furthermore, TTCH content is extremely important and decreases with decreasing seed vigor [45,46].
In this study, the soluble sugar content, conductivity, and MDA content of hard seeds were lower than those of non-hard seeds; this may be due to the damage of the membrane structure caused by the pre-imbibed seeds, which increased the extravasation of soluble sugar and other substances. However, the hard seeds have less extravasation of inclusions because the seed coat is hard and dense and the membrane structure is protected. The TTCH content and SOD activity of O. hosiei hard seeds were higher than those of non-hard seeds, which shows that the hard seeds of O. hosiei had fully matured, and the vigor was higher than that of the non-hard seeds. The changes in physiological metabolism during the soaking process led to higher TTCH content in hard seeds. The small water uptake rate and long imbibition time of the hard seeds of O. hosiei resulted in a large amount of active oxygen in the seed cells, leading to an increase in SOD activity.

5. Conclusions

The seed coat is the main obstacle to absorbing water of O. hosiei seeds, the main reason being that the seed coat is composed of cuticle, palisade cells, and thick-walled cells with impermeability. The dormancy caused by the seed coat barrier could be broken by hot water and acid etching treatment, and the effect of breaking O. hosiei hard seed dormancy by concentrated hot water treatment was better than that by acid treatment. The hard seeds had high TTCH content and SOD activity, and the membrane lipid peroxidation was relatively slight, which helped to maintain the integrity of the cell membrane, and the extravasation was less, so the germination rate and vigor index were also higher.

Author Contributions

Conceptualization, X.W. and L.D.; validation, X.W. and L.D.; formal analysis, L.D.; investigation, L.D.; resources, X.W.; data curation, L.D. and Y.C.; writing—original draft preparation, L.D.; visualization, L.D.; supervision, X.W.; project administration, X.W.; funding acquisition, X.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been supported by the Department of Science and Technology of Guizhou Province. The research projects are “Guizhou Province High-level Innovative Talents Training Plan Project” and “Science And Technology Support Plan Of Guizhou Province, China”. The funding numbers are, respectively, [(2016) 5661] and [(2021) Genera 501].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 13 01077 g001 550
Figure 1. The water absorption curve of O. hosiei seeds. Among them, the intact seeds and scarified seeds were soaked, respectively, and then the degree of seed imbibition was determined.
Figure 1. The water absorption curve of O. hosiei seeds. Among them, the intact seeds and scarified seeds were soaked, respectively, and then the degree of seed imbibition was determined.
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Figure 2. Cross section of O. hosiei seed coat by electron microscope scanning. (A–C) are the cross sections of untreated seeds, hot-water-treated seeds, and acid-treated seeds of O. hosiei, respectively.
Figure 2. Cross section of O. hosiei seed coat by electron microscope scanning. (A–C) are the cross sections of untreated seeds, hot-water-treated seeds, and acid-treated seeds of O. hosiei, respectively.
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Figure 3. Longitudinal sections of O. hosiei seed coat by electron microscope scanning. (A–C) are the longitudinal sections of untreated seeds, hot-water-treated seeds, and acid-treated seeds of O. hosiei, respectively.
Figure 3. Longitudinal sections of O. hosiei seed coat by electron microscope scanning. (A–C) are the longitudinal sections of untreated seeds, hot-water-treated seeds, and acid-treated seeds of O. hosiei, respectively.
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Agriculture 13 01077 g004 550
Figure 4. Comparison of electrical conductivity between non-hard seeds and hard seeds. The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
Figure 4. Comparison of electrical conductivity between non-hard seeds and hard seeds. The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
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Agriculture 13 01077 g005 550
Figure 5. Comparison of biochemical substance content of different seeds. (A) Changes in soluble sugar content; (B) changes in malondialdehyde content; (C) changes in dehydrogenase activity; (D) changes in superoxide dismutase activity. The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
Figure 5. Comparison of biochemical substance content of different seeds. (A) Changes in soluble sugar content; (B) changes in malondialdehyde content; (C) changes in dehydrogenase activity; (D) changes in superoxide dismutase activity. The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
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Table
Table 1. Comparison of germination indexes and seedling root vigor by the different treatments.
Table 1. Comparison of germination indexes and seedling root vigor by the different treatments.
TreatmentTimeGermination Ratio (%)Germination Potential (%)Germination IndexRoot Vigor (mg/g)
H2SO45 min60 ± 0.57 cC31 ± 0.57 dD12 ± 1.00 dD2.34 ± 0.01 eD
10 min63 ± 1.15 cC31 ± 0.00 dCD13 ± 0.57 cdCd2.38 ± 0.01 dCD
15 min63 ± 0.57 cC33 ± 0.00 cC14 ± 0.58 cC2.41 ± 0.01 cdCD
20 min82 ± 0.00 aA51 ± 0.58 aA25 ± 0.58 aA2.64 ± 0.01 bcC
25 min77 ± 1.15 bB45 ± 1.15 bB21 ± 0.58 bB2.51 ± 0.01 bB
30 min51 ± 0.58 dD25 ± 0.58 eE9 ± 0.58 eE2.13 ± 0.01 fE
CK36 ± 0.00 eE11 ± 0.57 fF7 ± 0.58 fF2.14 ± 0.02 fE
Hot water70 °C56 ± 1.00 dD26 ± 1.52 dD10 ± 0.58 dD2.15 ± 0.02 dD
80 °C67 ± 0.58 cC34 ± 0.58 cC15 ± 0.58 cC2.38 ± 0.01 cC
90 °C81 ± 0.58 bB49 ± 0.58 bB23 ± 0.58 bB3.10 ± 0.02 bB
100 °C86 ± 0.58 aA58 ± 0.58 aA28 ± 0.58 aA3.3 ± 0.02 aA
CK36 ± 0.00 eE11 ± 0.58 eE7 ± 0.58 eE2.14 ± 0.02 dD
Note: The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
Table
Table 2. Comparison of biochemical substance content and seed coat permeability of hard seed by the different etching treatments and hot water treatments.
Table 2. Comparison of biochemical substance content and seed coat permeability of hard seed by the different etching treatments and hot water treatments.
TreatmentTimeConductivity (μs/cm·g)SSC (mg/g)TTCH Activity (μg/g)SOD Activity (U/g)MDA Content (nmol/g)
H2SO45 min26.19 ± 0.93 cB20.60 ± 0.71 abA2.89 ± 0.02 cBC155.94 ± 4.48 dC6.38 ± 0.22 cC
10 min26.10 ± 1.36 cB20.48 ± 1.07 abA2.99 ± 0.04 bB198.35 ± 1.09 cB6.55 ± 0.09 cC
15 min26.50 ± 1.25 cB20.39 ± 0.91 bA2.80 ± 0.02 dC212.59 ± 2.19 bA6.84 ± 0.10 cC
20 min26.35 ± 2.16 cB20.17 ± 0.97 bA2.85 ± 0.01 cdC223.70 ± 2.49 aA7.37 ± 0.42 cC
25 min32.50 ± 0.84 bA22.12 ± 0.94 abA2.23 ± 0.02 eD89.40 ± 1.97 eD9.37 ± 0.48 bB
30 min36.38 ± 0.86 aA22.80 ± 0.84 aA2.15 ± 0.02 eD79.86 ± 2.77 fD11.16 ± 0.63 aA
CK19.27 ± 0.90 dC16.68 ± 0.45 cB3.14 ± 0.05 aA160.72 ± 5.31 dC3.43 ± 0.34 dD
Hot water70 °C21.67 ± 0.13 aA17.44 ± 1.60 aA4.35 ± 0.00 aA234.05 ± 2.61 aA6.84 ± 0.10 cC
80 °C22.17 ± 0.34 aA17.09 ± 0.04 aA3.85 ± 0.01 bB229.69 ± 1.67 aA7.37 ± 0.42 cC
90 °C22.70 ± 0.27 aA18.06 ± 0.42 aA3.72 ± 0.01 cC227.16 ± 2.14 aA9.37 ± 0.48 bB
100 °C22.81 ± 1.17 aA18.72 ± 1.12 aA3.70 ± 0.02 cC226.05 ± 2.13 aA11.16 ± 0.63 aA
CK19.27 ± 0.90 bB16.68 ± 0.45 aA3.14 ± 0.01 dD160.72 ± 5.31 bB3.43 ± 0.34 dD
Note: The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
Table
Table 3. Comparison of germination indexes and seedling root vigor between hard seeds and non-hard seeds.
Table 3. Comparison of germination indexes and seedling root vigor between hard seeds and non-hard seeds.
SampleGermination Ratio (%)Germination Potential (%)Germination IndexRoot Vigor (mg/g)
X124 ± 0.58 fF16 ± 1.00 fF6 ± 0.00 fF2.15 ± 0.03 eD
X229 ± 0.58 eE18 ± 0.58 fF7 ± 0.00 fF2.34 ± 0.03 deD
X340 ± 0.58 dD26 ± 0.58 eE9 ± 0.58 eE2.79 ± 0.02 cdCD
X445 ± 1.00 cC32 ± 1.00 dD13 ± 0.58 dD3.13 ± 0.03 bcBC
X546 ± 0.57 cC35 ± 0.00 cC15 ± 0.58 cC3.44 ± 0.02 bBC
X651 ± 0.58 bB41 ± 0.58 bB19 ± 0.58 bB3.64 ± 0.60 bB
H75 ± 0.58 aA64 ± 1.00 aA24.00 ± 0.00 aA4.67 ± 0.02 aA
Note: The different capital letters show significant difference at 0.01 level; the different lowercase letters show significant difference at 0.05 level.
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Dai, L.; Chen, Y.; Wei, X. Hard Seed Characteristics and Seed Vigor of Ormosia hosiei. Agriculture 2023, 13, 1077. https://doi.org/10.3390/agriculture13051077

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Dai L, Chen Y, Wei X. Hard Seed Characteristics and Seed Vigor of Ormosia hosiei. Agriculture. 2023; 13(5):1077. https://doi.org/10.3390/agriculture13051077

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Dai, Li, Yanwei Chen, and Xiaoli Wei. 2023. "Hard Seed Characteristics and Seed Vigor of Ormosia hosiei" Agriculture 13, no. 5: 1077. https://doi.org/10.3390/agriculture13051077

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